Human Prostate Cancer Metastasis Research Articles

Modeling Human Prostate Cancer Metastasis in Mice via Resection of Subcutaneous Allografts.

The 5-year survival rate for patients diagnosed with distant metastatic prostate cancer in the United States is 30.6%. Therefore, there is a great need to develop in vivo model systems to study prostate cancer metastasis and to test potential therapeutics. Most murine prostate cancer metastatic models involve intracardiac or intraosseous implantation of cancer cells, which bypass the early stages of tumor cell migration and invasion. Herein we provide a detailed protocol for a novel method of resecting subcutaneous prostate cancer allografts in immunocompetent mice to produce spontaneous metastases and describe a pilot study using this method of tumor resection. Intact male FVB/NCrl mice (n = 9) were inoculated subcutaneously with Myc-CaP cells. Tumors were surgically resected, and mice were monitored for tumor recurrence. Animals were euthanized or died, and a full set of tissues was collected for histopathologic examination. Tumors took an average of 44 days (range 23–61) to reach 1.7 cm in any direction. All tumors were resectable, and resection of the tumors increased the study length by 70 days (range 30–121). One mouse was euthanized early of an unrelated cause, and of eight remaining mice, four developed tumor recurrence at the site of resection. One mouse developed bone metastases, one mouse developed metastases to the abdominal cavity, and two mice showed signs of local invasion. This study demonstrates that resection of subcutaneous Myc-CaP cell allografts in mice results in local tumor recurrence and the development of distant metastases, providing a new model system to study prostate cancer metastasis in vivo.

Carbon sources and pathways for citrate secreted by human prostate cancer cells determined by NMR tracing and metabolic modeling

Prostate epithelial cells have the unique capacity to secrete large amounts of citrate, but the carbon sources and metabolic pathways that maintain this production are not well known. We mapped potential pathways for citrate carbons in the human prostate cancer metastasis cell lines LNCaP and VCaP, for which we first established that they secrete citrate (For LNCaP 5.6 ± 0.9 nmol/h per 106 cells). Using 13C-labeled substrates, we traced the incorporation of 13C into citrate by NMR of extracellular fluid. Our results provide direct evidence that glucose is a main carbon source for secreted citrate. We also demonstrate that carbons from supplied glutamine flow via oxidative Krebs cycle and reductive carboxylation routes to positions in secreted citrate but likely do not contribute to its net synthesis. The potential anaplerotic carbon sources aspartate and asparagine did not contribute to citrate carbons. We developed a quantitative metabolic model employing the 13C distribution in extracellular citrate after 13C glucose and pyruvate application to assess intracellular pathways of carbons for secreted citrate. From this model, it was estimated that in LNCaP about 21% of pyruvate entering the Krebs cycle is converted via pyruvate carboxylase as an anaplerotic route at a rate more than sufficient to compensate carbon loss of this cycle by citrate secretion. This model provides an estimation of the fraction of molecules, including citrate, leaving the Krebs cycle at every turn. The measured ratios of 13C atoms at different positions in extracellular citrate may serve as biomarkers for (malignant) epithelial cell metabolism.

Prostate-specific membrane antigen modulates the progression of prostate cancer by regulating the synthesis of arginine and proline and the expression of androgen receptors and Fos proto-oncogenes

ABSTRACT The expression of prostate-specific membrane antigen (PSMA) is strikingly upregulated during oncogenesis and prostate cancer (PCa) progression, but the functions of this antigen in PCa remain unclear. Here, we constructed PSMA-knockdown LNCaP and 22rv1 cell lines and performed metabonomic and transcriptomic analyses to determine the effects of PSMA on PCa metabolism and transcription. The metabolism of arginine and proline was detected using specific kits. The mRNA and protein expression levels of the identified differentially expressed genes were quantified by RT-qPCR and Western blotting. The proliferation of each cell line was evaluated through CCK-8, EdU and colony formation assays. The migration and invasion abilities of each cell line were detected using wound healing and transwell assays, respectively. PSMA knockdown led to metabolic disorder and abnormal transcription in PCa and resulted in inhibition of the proliferation and metastasis of PCa cells in vitro and in vivo. The depletion of PSMA also promoted the biosynthesis of arginine and proline, inhibited the expression of AR and PSA, and induced the expression of c-Fos and FosB. PSMA plays an important role in the metabolism, proliferation and metastasis of human PCa and may be a promising therapeutic target.

MiR-622 acts as a tumor suppressor to induce cell apoptosis and inhibit metastasis in human prostate cancer.

Growing evidence indicating the critical modulator roles of microRNAs (miRNAs) involved in prostate cancer (PCa) metastasis that holds great promise as therapeutic targets. Herein, we transfected the miR-622 mimic into PC3 cells and evaluated the effects of this interference on these tumour cells' growth and the expression of specific metastatic genes. Transfecting of miR-622 mimic and inhibitor, negative control (NC) inhibitor and NC was established using Lipofectamine 2000. The mRNA levels of miR-622 and metastatic genes were evaluated using the qRT-PCR and Western blot. Cytotoxic effects of miR-622 were assessed by MTT. Apoptosis was detected using an ELISA cell death assay kit. miR-622 is down-regulated in PC3 cells. As expected, cell viability effects after transfection were described as miR-622 inhibitor >NC and NC inhibitor >miR-622 mimic (p<.01). Importantly, we showed that transfected miR-622 mimic could enhance the apoptosis of PC3 cells, while transfected miR-622 inhibitor could decrease cell apoptosis (p<.01). Furthermore, miR-622 overexpression could increase significantly down-regulated the MMP2, MMP9, CXCR-4, c-Myc and K-Ras expression levels. Findings demonstrate a novel mechanism by which miR-622 modulates PCa cells' metastasis by targeting metastatic genes. These data confirm the tumour-suppressive function of miR-622 in PCa cells by enhancing apoptosis and reducing metastasis.

Role of ZBTB7A zinc finger in tumorigenesis and metastasis.

The zinc finger and BTB (broad-complex, tramtrack and bric a brac) domain containing protein 7A (ZBTB7A) is a pleiotropic transcription factor that plays an important role in various stages of cell proliferation, differentiation, and other developmental processes. ZBTB7A is a member of the POK family that directly and specifically binds to short DNA recognition sites located near their target genes thereby acting as transcriptional activator or repressor. ZBTB7A overexpression has been associated with tumorigenesis and metastasis in various human cancer types, including breast, prostate, lung, ovarian, and colon cancer. However in some instances downregulation of ZBTB7A results in tumor progression, suggesting its role as a tumor suppressor. ZBTB7A is involved with complicated regulatory networks which include protein-protein and protein-nucleic acid interactions. ZBTB7A involvement in cancer progression and metastasis is perhaps enabled through the regulation of various signaling pathways depending on the type and genetic context of cancer. The association of ZBTB7A with other proteins affects cancer aggressiveness, therapeutic resistance and clinical outcome. This review focuses on the involvement of ZBTB7A in various signaling pathways and its role in cancer progression. We will also review the literature on ZBTB7A and cancer which could be potentially explored for its therapeutic implications.

Spirogyra neglecta (Hassall) Kützing attenuates metastasis of castration-resistant human prostate cancer via the blockage of AKT signaling pathway

Spirogyra neglecta (Hassall) Kützing, a filamentous fresh water green alga, has clearly demonstrated its anti-inflammation properties; however, the anti-metastasis activity of this plant has not yet been elucidated. This study aimed to investigate the anti-metastasis effects of S. neglecta against castration-resistant prostate cancer cells. Transwell assays were used to determine the anti-metastasis properties of S. neglecta against PC3 cells. Furthermore, the metabolic profiling, 1H-NMR based metabolomics analysis, was conducted to identify the potential active compounds. The results revealed that S. neglecta ethanolic extract (SnEE) significantly inhibited PC3 cells invasion and migration. The metabolomics for the profiling-based screening of very well-known medicinal herbs in Asian countries (S. neglecta, S. platensis, T. erecta, C. siamea) have indicated a significant positive association between gallic acid, gentisic acid and the anti-metastasis properties of the SnEE. Thus, the solvent-partition technique was performed to concentrate the phenolic compounds in SnEE and then to establish the S. neglecta ethyl acetate fraction (SnEA). Mechanistically, SnEA inhibited PC3 cells metastasis through the inhibition of MMP-9 secretion, epithelial-mesenchymal transition, and the Akt signaling pathway (p<0.01). Our study, for the first time, showed that S. neglecta extract displayed remarkable anti-metastasis properties against castration-resistant prostate cancer by inhibiting the Akt signaling pathway. These findings appeared to support the use of S. neglecta extract as a natural supplement in the effective treatment of prostate cancer.

Hard antler extract inhibits invasion and epithelial–mesenchymal transition of triple-negative and Her-2+ breast cancer cells by attenuating nuclear factor-κB signaling

Ethnopharmacological relevanceHard antler extract (HAE) is a traditional Chinese medicine and has potent antitumor, antioxidative, anti-inflammatory, and immunomodulatory activities. Previous studies have demonstrated that HAE can inhibit human prostate cancer metastasis and murine breast cancer proliferation. However, the effect of HAE on human breast cancer cells has not been clarified.Aim of the study: To investigate the effects and underlying mechanism of HAE on self-renewal of stem-like cells and spontaneous and transforming growth factor (TGF)-β1-enhanced wound healing, invasion and epithelial–mesenchymal transition (EMT) in breast cancer cells. MethodsHAE was prepared from sika deer by sequential enzymatic digestions and the active compounds were determined by HPLC. The effects of HAE on the viability, mammosphere formation, wound healing and invasion of MDA-MB-231 and SK-BR3 cells were determined. The impact of HAE treatment on spontaneous and TGF-β1-promoted EMT and the nuclear factor (NF)-κB signaling in breast cancer cells was examined by quantitative RT-PCR and western blotting. ResultsTreatment with HAE at varying concentrations did not change the viability of breast cancer cells. However, HAE at 0.25 or 0.5 mg/mL significantly reduced the number and size of formed mammospheres, and inhibited spontaneous and TGF-β1-enhanced wound healing, invasion and EMT in MDA-MB-231 and SK-BR3 cells in a dose-dependent manner. TGF-β1 treatment significantly decreased IκBα expression and increased NF-kBp65 phosphorylation in breast cancer cells, indicating that TGF-β1 enhanced NF-κB signaling. In contrast, HAE treatment attenuated the spontaneous and TGF-β1-enhanced NF-κB signaling in breast cancer cells. ConclusionOur data indicated that HAE inhibited the self-renewal of stem-like cells and spontaneous and TGF-β1-enhanced wound healing, invasion and EMT in breast cancer cells by attenuating the NF-κB signaling in vitro.

Inhibition of TMPRSS2 by HAI-2 reduces prostate cancer cell invasion and metastasis.

TMPRSS2 is an important membrane-anchored serine protease involved in human prostate cancer progression and metastasis. A serine protease physiologically often comes together with a cognate inhibitor for execution of proteolytically biologic function; however, TMPRSS2’s cognate inhibitor is still elusive. To identify the cognate inhibitor of TMPRSS2, in this study, we applied co-immunoprecipitation and LC/MS/MS analysis and isolated hepatocyte growth factor activator inhibitors (HAIs) to be potential inhibitor candidates for TMPRSS2. Moreover, the recombinant HAI-2 proteins exhibited a better inhibitory effect on TMPRSS2 proteolytic activity than HAI-1, and recombinant HAI-2 proteins had a high affinity to form a complex with TMPRSS2. The immunofluorescence images further showed that TMPRSS2 was co-localized to HAI-2. Both KD1 and KD2 domain of HAI-2 showed comparable inhibitory effects on TMPRSS2 proteolytic activity. In addition, HAI-2 overexpression could suppress the induction effect of TMPRSS2 on pro-HGF activation, extracellular matrix degradation and prostate cancer cell invasion. We further determined that the expression levels of TMPRSS2 were inversely correlated with HAI-2 levels during prostate cancer progression. In orthotopic xenograft animal model, TMPRSS2 overexpression promoted prostate cancer metastasis, and HAI-2 overexpression efficiently blocked TMPRSS2-induced metastasis. In summary, the results together indicate that HAI-2 can function as a cognate inhibitor for TMPRSS2 in human prostate cancer cells and may serve as a potential factor to suppress TMPRSS2-mediated malignancy.

MEIS-mediated suppression of human prostate cancer growth and metastasis through HOXB13-dependent regulation of proteoglycans.

The molecular roles of HOX transcriptional activity in human prostate epithelial cells remain unclear, impeding the implementation of new treatment strategies for cancer prevention and therapy. MEIS proteins are transcription factors that bind and direct HOX protein activity. MEIS proteins are putative tumor suppressors that are frequently silenced in aggressive forms of prostate cancer. Here we show that MEIS1 expression is sufficient to decrease proliferation and metastasis of prostate cancer cells in vitro and in vivo murine xenograft models. HOXB13 deletion demonstrates that the tumor-suppressive activity of MEIS1 is dependent on HOXB13. Integration of ChIP-seq and RNA-seq data revealed direct and HOXB13-dependent regulation of proteoglycans including decorin (DCN) as a mechanism of MEIS1-driven tumor suppression. These results define and underscore the importance of MEIS1-HOXB13 transcriptional regulation in suppressing prostate cancer progression and provide a mechanistic framework for the investigation of HOXB13 mutants and oncogenic cofactors when MEIS1/2 are silenced.

A Multifunctional Therapy Approach for Cancer: Targeting Raf1- Mediated Inhibition of Cell Motility, Growth, and Interaction with the Microenvironment.

Prostate cancer cells move from their primary site of origin, interact with a distant microenvironment, grow, and thereby cause death. It had heretofore not been possible to selectively inhibit cancer cell motility. Our group has recently shown that inhibition of intracellular activation of Raf1 with the small-molecule therapeutic KBU2046 permits, for the first time, selective inhibition of cell motility. We hypothesized that simultaneous disruption of multiple distinct functions that drive progression of prostate cancer to induce death would result in advanced disease control. Using a murine orthotopic implantation model of human prostate cancer metastasis, we demonstrate that combined treatment with KBU2046 and docetaxel retains docetaxel's antitumor action, but provides improved inhibition of metastasis, compared with monotherapy. KBU2046 does not interfere with hormone therapy, inclusive of enzalutamide-mediated inhibition of androgen receptor (AR) function and cell growth inhibition, and inclusive of the ability of castration to inhibit LNCaP-AR cell outgrowth in mice. Cell movement is necessary for osteoclast-mediated bone degradation. KBU2046 inhibits Raf1 and its downstream activation of MEK1/2 and ERK1/2 in osteoclasts, inhibiting cytoskeleton rearrangement, resorptive cavity formation, and bone destruction in vitro, with improved effects observed when the bone microenvironment is chemically modified by pretreatment with zoledronic acid. Using a murine cardiac injection model of human prostate cancer bone destruction quantified by CT, KBU2046 plus zoledronic exhibit improved inhibitory efficacy, compared with monotherapy. The combined disruption of pathways that drive cell movement, interaction with bone, and growth constitutes a multifunctional targeting strategy that provides advanced disease control.

Novel human Ab against vascular endothelial growth factor receptor 2 shows therapeutic potential for leukemia and prostate cancer.

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor‐associated endothelial cells, where it modulates tumor‐promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti‐VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti‐VEGFR2‐AF, which shows excellent VEGFR2 binding activity. Anti‐VEGFR2‐AF bound Ig‐like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF‐A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti‐VEGFR2‐AF inhibited capillary structure formation and exerted Ab‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC‐3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC‐3 xenograft mouse model, treatment with anti‐VEGFR2‐AF repressed tumor growth and angiogenesis as effectively and safely as US FDA‐approved anti‐VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti‐VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL‐60 human leukemia‐xenografted mice, anti‐VEGFR2‐AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti‐VEGFR2‐AF has strong potential as a cancer therapy that could directly target VEGFR2‐expressing tumor cells in addition to its anti‐angiogenic action.

The role of WNT10B in normal prostate gland development and prostate cancer

WNT signaling is implicated in embryonic development, and in adult tissue homeostasis, while its deregulation is evident in disease. This study investigates the unique roles of canonical WNT10B in both normal prostate development and prostate cancer (PCa) progression. Organ culture and rat ventral prostates (VPs) were used to study Wnt10b ontogeny and growth effect of WNT10B protein. PB-SV40 LTag rat VPs were utilized for Wnt expression polymerase chain reaction (PCR) array and immunohistochemistry. Human localized PCa tissue microarrays (TMAs) were investigated for differential WNT10B expression. Human RNA-seq data sets were queried for differential expression of WNT10B in metastatic and localized PCa. Knockdown of WNT10B in PC3 cells was utilized to study its effects on proliferation, stemness, epithelial to mesenchymal transition (EMT), and xenograft propagation. Wnt10b expression was highest at birth and rapidly declined in the postnatal rat VP. Exogenous WNT10B addition to culture developing VPs decreased growth suggesting an antiproliferative role. VPs from PB-SV40 LTag rats with localized PCa showed a 25-fold reduction in Wnt10b messenger RNA (mRNA) expession, confirmed at the protein level. Human PCa TMAs revealed elevated WNT10B protein in prostate intraepithelial neoplasia compared with normal prostates but reduced levels in localized PCa specimens. In contrast, RNA-seq data set of annotated human PCa metastasis found a significant increase in WNT10B mRNA expression compared with localized tumors suggesting stage-specific functions of WNT10B. Similarly, WNT10B mRNA levels were increased in metastatic cell lines PC3, PC3M, as well as in HuSLC, a PCa stem-like cell line, as compared with disease-free primary prostate epithelial cells. WNT10B knockdown in PC3 cells reduced expression of EMT genes, MMP9 and stemness genes NANOG and SOX2 and markedly reduced the stem cell-like side population. Furthermore, loss of WNT10B abrogated the ability of PC3 cells to propagate tumors via serial transplantation. Taken together, these results suggest a dual role for WNT10B in normal development and in PCa progression with opposing functions depending on disease stage. We propose that decreased WNT10B levels in localized cancer allow for a hyperproliferative state, whereas increased levels in advanced disease confer a stemness and malignant propensity which is mitigated by knocking down WNT10B levels. This raises the potential for WNT10B as a novel target for therapeutic intervention in metastatic PCa.

Abstract 4523: A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization

Abstract Background: Bone metastasis in human prostate cancer remains a major clinical problem since no effective therapy exists. The RANKL/RANK pathway plays a predominant role in the interaction between metastasized prostate cancer cells and osteoclasts that increases the bone turnover. The current therapies, including targeting RANKL with denosumab, address the growth of prostate tumor cells that have already colonized the bone, but are largely ineffective in prolonging the survival of human prostate cancer patients with bone metastasis. Further, a major impediment to prostate cancer bone metastasis research is the lack of an animal model that spontaneously recapitulates human prostate cancer bone metastasis in the context of an intact immune system. Results: To overcome this major limitation, we have developed a novel syngeneic mouse model to study prostate cancer bone metastasis. Both the CXCL12/CXCR4 and RANKL/RANK pathways have been reported to be overexpressed / dysregulated in human prostate cancer bone metastatic samples. Data generated utilizing our immune-intact mouse model shows that the CXCL12/CXCR4 and RANKL/RANK pathways co-operate with each other to drive prostate cancer bone metastasis. Studies have shown that targeting the CXCL12/CXCR4 and RANKL/RANK pathways individually affects the immune system, thereby making our syngeneic mouse model an indispensable tool for studying the critical co-operation between these 2 pathways in the manifestation of human prostate cancer bone metastasis. Extending our earlier findings that RANKL drives PCa metastases in immune-deficient mice (Chu et al., 2014) to immune-intact C57/Bl6 mice, we found that MPC3 mouse PCa cells with RANKL overexpression (MPC3-Luc-GFP-RANKL) develop 70-80% limb and jaw within 4 weeks of intra-cardiac injection in these syngeneic mice. Control MPC3 cells had no bone metastasis. Bone lesions visualized by luciferase imaging and X-ray were confirmed by micro CT and immunohistochemistry. RANKL signaling drove bone and visceral metastases via the downstream CXCL12/CXCR4 signaling axis. MPC3-Luc-GFP-RANKL cells showed increased CXCR4 protein levels by immunohistochemistry. Metastatic bone marrow flush showed dramatically increased levels of CXCL12 mRNA compared with control mice (MPC3-Luc-GFP-EV). Conclusion: In sum, 1) circulating PCa cells induce a marked CXCL12 elevation after colonizing bone, triggering chemotaxis and recruiting CXCR4-positive PCa cells to migrate to bone; and 2) osteomimetic PCa cells with increased RANKL expression interact with osteoclasts to enhance bone resorption and turnover, releasing additional growth factors and chemokines for PCa cell growth and survival in bone. Citation Format: Srinivas Nandana, Murali Gururajan, Manisha Tripathi, Chia-Yi Chu, Haiyen Zhau, Stephen Shiao, Leland Chung. A novel syngeneic mouse model of prostate cancer bone metastasis: Mechanisms of chemotaxis and bone colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4523.

Human Sinusoidal Subendothelial Cells Regulate Homing and Invasion of Circulating Metastatic Prostate Cancer Cells to Bone Marrow.

Subendothelial cells (pericytes) are the clonogenic, multipotent and self-renewing skeletal stem cells (SSCs) found in bone marrow (BM) stroma. They express genes maintaining hematopoietic stem cell (HMC) niche identity and, transplanted in immunocompromised mice, organize the hematopoietic microenvironment (HME) generating humanized bone/BM ossicles. To create a mouse model of hematogenous metastasis of human prostate cancer (PC) cells to human bone/BM, we injected PC cells in the blood circulatory system of Severe Combined Immunodeficiency (SCID)/beige mice bearing heterotopic ossicles. Results indicate that PC cells could efficiently home to mice-implanted extraskeletal BM ossicles, but were not able to colonize mice skeletal segments. In humanized bone/BM ossicles, early foci of PC cells occupied a perisinusoidal position, in close contact with perivascular stromal cells. These findings demonstrate the importance of the SSC compartment in recreating a suitable environment to metastatic PC cells. Our data support the hypothesis that BM SSCs committed to a pericyte fate can specify for homing niches of PC cells, suggesting an involvement of specific interactions with subendothelial stromal cells in extravasation of circulating metastatic PC cells to BM.

Role of exosomal small RNA in prostate cancer metastasis.

Prostate cancer (PCa) is the second most common cancer in men worldwide. When the disease becomes metastatic, limited treatment strategies exist, and metastatic disease prognoses are difficult to predict. Recently, evidence has emerged, which indicates that small RNAs are detectable in patient fluids, and exosomal small RNA ectopic expression is correlated with the development, progression, and metastasis of human PCa; however, the role of small RNAs in PCa is only partially understood. In this review, we discuss the research status regarding circulating exosomal small RNAs and applications using these small RNAs in PCa particularly looking at metastatic disease. Exosomal small RNAs could be used as potential biomarkers for the early diagnosis, micrometastasis detection, and prognosis of PCa.

PO-179 Breast cancer cells and macrophages in a paracrine-juxtacrine loop

IntroductionCancer metastasis is a main cause for mortality. Dysregulation of pericellular proteolysis usually accounts for cancer cell invasion and metastasis. In this study, we are interested in delineating the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate and lung cancer cell invasion and metastasis.Material and methodsWe used different prostate and lung cancer cells and animal models to examine the role of HAI-2 in prostate and lung cancer cell invasion and metastasis.Results and discussionsIn this study, we identified a membrane-anchored serine protease inhibitor, hepatocyte growth factor activator inhibitor-2 (HAI-2), was down-regulated in human prostate and lung cancer. The decreased levels of HAI-2 were related to the progression of human prostate and lung adenocarcinoma. HAI-2 overexpression can repress both cancer cell migration and invasion. Recombinant HAI-2 proteins can inhibit both cancer cell motility. In addition, HAI-2 overexpression reduced the tumour growth and metastasis of prostate cancer cells, while down-regulation of HAI-2 can increase the metastatic ability of lung adenocarcinoma cells in xenografted animal models.ConclusionHAI-2 functions as a suppressor to inhibit the cell invasion and metastasis of human prostate and lung cancer.

PO-176 Hepatocyte growth factor activator inhibitor-2 suppresses human prostate and lung cancer cell invasion and metastasis

IntroductionIntegrins are glycosylated transmembrane receptor family proteins, mediate cancer cell to extracellular matrix (ECM) interaction. Integrins are essential for adhesion and migration of cancer cells and frequently associate with poor disease outcome. In addition, glycosylation of integrins modulates receptor and ligand binding affinity. However, the physiological role of glycosylation of integrins remain unclear. Based on our recent findings that disruption of glycosphingolipids triggering loss of α2,6-sialylation Alam et al. 2017 here, we identified integrin α2 as candidate and investigated its potential role in facilitating ovarian cancer metastasis.Material and methodsHypersialylated proteins were identified by lectin-enrichment proteomic analysis. CRISPR-Cas9 mediated site-specific deletion of integrin α2 together with a lentiviral-mediated rescue system were established in four ovarian cancer cell lines (IGROV1, SKOV3, SKOV3ip, and OVCAR4). ECM- and mesothelial cell- adhesion assays were applied to evaluate integrin-mediated in vitro cell adhesion, while a zebrafish transplant model was used to study in vivo cell invasion ability. Finally, integrin expression and sialylation profile were determined in patient-derived matched primary and metastatic ovarian cancer tumour specimens.Results and discussionsProteomic analysis identified integrin α2 as a hypersialylated protein in ovarian cancer cells. Loss of sialylation on N-glycoproteins of cancer cells reduced cell adhesion to collagen type I, fibronectin, and laminin. Furthermore, integrin α2 knockout cancer cells abolished cell adhesion to collagen whereas rescuing integrin α2 expression in those knockout cells fully restored the adhesion ability. We also identified an elevated level of intergin α2 in patient-derived metastatic tumour compared to primary site and tumor-free omentum, indicating a niche for ovarian cancer cells toward omental metastasis.ConclusionOur data demonstrate that α2,6 sialylation on integrin α2 triggers ovarian cancer cell adhesion to metastatic sites. Therefore, blocking sialylation and integrin α2 may be a therapeutic target for preventing ovarian cancer metastasis in the future.

SGK1 inhibition-induced autophagy impairs prostate cancer metastasis by reversing EMT

BackgroundDespite SGK1 has been identified and characterized as a tumor-promoting gene, the functions and underlying mechanisms of SGK1 involved in metastasis regulation have not yet been investigated in cancer.MethodsWe investigated the cellular responses to GSK650394 treatment and SGK1 silencing (or overexpression) in human prostate cancer (PCa) cell lines and PC3 xenografts by wound healing assay, migration and invasion assay, western blotting, immunofluorescence and immunohistochemistry.ResultsIn the present study, we found that SGK1 expression positively correlates with human prostate cancer (PCa) progression and metastasis. We show that SGK1 inhibition significantly attenuates EMT and metastasis both in vitro and in vivo, whereas overexpression of SGK1 dramaticlly promoted the invasion and migration of PCa cells. Our further results suggest that SGK1 inhibition induced antimetastatic effects, at least partially via autophagy-mediated repression of EMT through the downregulation of Snail. Moreover, ectopic expression of SGK1 obviously attenuated the GSK650394-induced autophagy and antimetastatic effects. What’s more, dual inhibition of mTOR and SGK1 enhances autophagy and leads to synergistic antimetastatic effects on PCa cells.ConclusionsTaken together, this study unveils a novel mechanism in which SGK1 functions as a tumor metastasis-promoting gene and highlights how co-targeting SGK1 and autophagy restrains cancer progression due to the amplified antimetastatic effects.

MiR-221-5p enhances cell proliferation and metastasis through post-transcriptional regulation of SOCS1 in human prostate cancer

BackgroundTo investigate the effect of miR-221-5p on cell proliferaton and metastasis of human prostate cancer in vitro and vivo.MethodsWe established PC3 cell lines with stable overexpression or silencing of miRNA-221-5p via lentivirus infection. miRNA-221-5p and its target gene SOCS1 expression levels in the stable cells were analyzed by real-time polymerase chain reaction (RT-PCR) and western blotting. Using luciferase reporter assays to study the relationship between miR-221-5p and SOCS1. Cell proliferative activity was measured using the MTT assay and colony formation assay. Migration ability was assessed using wound-healing assay and transwell assay. To further study the function of miR-221-5p in human prostate cancer we established nude mice xenograft model in vivo.ResultsmiR-221-5p regulates the proliferation, migration of prostate cancer cells in vitro and tumorigenesis in vivo by regulating socs1 expression through targeted its 3’UTR, and miR-221-5p regulates MAPK/ERK signaling pathway and EMT features in prostate cancer cells.ConclusionsUp-regulation and silencing of miR-221-5p expression in prostate cancer cells are correlated with cell proliferation, migration and tumorigenesis, which suggest that miR-221-5p plays an important role in prostate cancer progression.

Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate

Backgroundc-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines.ResultsEleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive.ConclusionThe present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.